Cartilage is critical to skeletal development, growth and function. The cartilage collagen fibril which is comprised of types II, IX and XI collagen is responsible for the form and integrity of cartilage. Compared to collagen fibrils in other connective tissues, much less is known about cartilage collagen fibrils. Indeed, many questions remain unresolved about their organization and assembly, the function of minor fibril constituents, the role of the cell in regulating fibril formation, etc. We propose to use a combined genetic, cellular and molecular biology approach and a unique group of naturally occurring human mutants of chondrogenesis to address these and related questions. Studies over the past 5 years have identified a group of human chondrodysplasias called Dominant Cartilage Matrix Fibril Dysplasias (DCMFDs) which share dominant inheritance and qualitatively similar clinical and pathologic phenotypes. The latter includes structural abnormalities of cartilage collagen fibrils, electrophoretic abnormalities of cartilage collagens and enlargement of RER. We hypothesize that DCMFDs result from heterozygous mutations of the genes encoding the constituent chains of types II, IX and XI collagens which alter biosynthetic processing of the molecules and fibrillogenesis leading to a defective cartilage matrix. The goal of the continuation of this project is to characterize a spectrum of DCMFD mutations and examine their effects on chondrogenesis to elucidate the molecular basis of these disorders and their phenotypic variability and gain insight into normal cartilage collagen fibril biology. The analyses of each of over 40 DCMFD cases will be done in a stepwise fashion centered around a chondrocyte culture system developed by the PI which provides cartilage-like tissue, newly synthesized collagens, DNA and RNA for analysis. Analysis of collagens will identify "candidate" collagen genes for further study. Southern and Northern blot analyses of genomic DNA and mRNA will then be used to screen candidate genes for gross mutations. To detect the mire likely subtle mutations, cDNA will be synthesized from total cellular RNA. Sequences of interest will then be amplified by PCR and analyzed by chemical cleavage to detect differences (possible mutations) between the candidate and normal cDNAs. "Suspect" cDNAs will then be sequenced. Once a mutation is identified and confirmed in genomic DNA, the mutant gene product will be further characterized biochemically; and the adverse effects of the mutation on collagen biosynthetic processing and fibril structure and organization will be investigated in vitro using pulse-chase, precursor localization, immunoelectron microscopic and rotary shadowing techniques. As mutations become known and mutation:phenotype correlations become possible, short segments of genomic DNA from formalin-fixed tissues of other DCMFD patients will be amplified by PCR to confirm the correlations and better define the molecular basis of the phenotypic variability. 10-15 mutations are expected to be defined.